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Development of Biological Control of Ralstonia solanacearum Through Antagonistic Microbial Populations

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Abstract

Potential antagonists were screened out and evaluated in-vitro and in-vivo as bio-control agents against the bacterial wilt pathogen. All the bio-control agents tested reduced the bacterial wilt disease to various degrees. Among the bio-control agents tested, EM instant was the most effective, while the degree of disease suppression by other microbes varied with the time of application. The effects of EM-FPE and Bokashi were not different from EM instant. Among the antagonists isolated from infested soils LR 10 showed a higher potential of disease suppression. The antagonists isolated from EM sources were effective to suppress against bacterial wilt pathogen. It means that EM contains some microbes, which can suppress the disease. Application method of bio-control agent should be suitable for antagonist's action and prior to pathogen attack.
INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY
1560–8530/2006/08–5–657–660
http://www.fspublishers.org
Development of Biological Control of Ralstonia solanacearum
Through Antagonistic Microbial Populations
MYINT LWIN
1
AND S.L. RANAMUKHAARACHCHI
Agricultural Systems and Engineering Program, Asian Institute of Technology, Pathumthani-12120, Thailand
1
Corresponding author’s e-mail: lwin399@gmail.com
ABSTRACT
Potential antagonists were screened out and evaluated in-vitro and in-vivo as bio-control agents against the bacterial wilt
pathogen. All the bio-control agents tested reduced the bacterial wilt disease to various degrees. Among the bio-control agents
tested, EM instant was the most effective, while the degree of disease suppression by other microbes varied with the time of
application. The effects of EM-FPE and Bokashi were not different from EM instant. Among the antagonists isolated from
infested soils LR 10 showed a higher potential of disease suppression. The antagonists isolated from EM sources were
effective to suppress against bacterial wilt pathogen. It means that EM contains some microbes, which can suppress the
disease. Application method of bio-control agent should be suitable for antagonist’s action and prior to pathogen attack.
Key Words: Biological control; Antagonist; Effective microorganisms (EM); Ralstonia solanacearum; Bacterial wilt
INTRODUCTION
Bacterial wilt, caused by Ralstonia solanacearum E.
Yabuuchi formerly known as Pseudomonas solanacearum
E.F. Smith is one of the most devastating, important and
wide-spread bacterial diseases of crops in tropical
environments (He et al., 1983). The pathogen has a wide
host range representing 44 families (He et al., 1983). Highly
susceptible crops are potato, tomato, egg plant, chili, bell
pepper and peanut. The disease has limited both commercial
and domestic level production (Somodi et al., 1993).
As the disease is widely distributed, it has a wide host
range and is mainly soil-borne; it is difficult to control with
chemicals and cultural practices (Grimault et al., 1993).
There seems to be a shift to the idea that biological control
can have an important role in the management of bacterial
wilt (Akiew et al., 1993). Biological control strategies may
either help development of alternative management
measures or be integrated with other practices for effective
disease management at the field level. Biological control not
only suppresses the disease and increases the crop yield but
will be important in preventing the environmental pollution
due to pesticides.
Several microorganisms have been tried out with
variable success for biological control of bacterial wilt
(Shekhawat et al., 1993). Effective microorganisms (EM) is
a mixture of beneficial microorganisms, which can increase
the crop yield and also protect against plant pathogens
(Higa, 1999). It is a mixed culture of photosynthetic
bacteria, Azotobacter, Streptomyces and Lactobacillus spp.,
which improve crop yield by increasing photosynthesis,
nitrogen fixation, controlling soil diseases and accelerating
decomposition of lignin material in the soil (Hussain et al.,
1993). EM have been reported to protect against
Pseudomonas bacteria (Castro et al., 1993).
Although the potential to suppress the pathogenic
organisms through biological means has been revealed,
sufficient information has not been generated so far to fully
support the development of biological control measures
against R. solanacearum. Therefore, the current study was
conducted to evaluate the effect of potential antagonists on
R. solanacearum in vivo and vitro and develop effective bio-
control measure.
MATERIALS AND METHODS
This study was conducted at the Agricultural system
laboratories of the Asian Institute of Technology, Thailand
during October 2004 to May 2005. The study included the
screening of potential antagonists and their evaluation in
vitro and vivo.
a) Screening of potential antagonists. Bacterial wilt
diseased tomato plant parts and infested soils were collected
from the farmers’ fields in the Pakchong district, Nakorn
Ratchasima province of the North-east Thailand where the
disease was prevalent. The pathogenic bacterium, R.
solanacearum was isolated from diseased tissue and
pathogenicity was confirmed by root dipped inoculation
method (Winstead & Kelman, 1952). Seven suspected
antagonists from bacterial disease infected soils and three
isolates from the commercially available EM preparations
were isolated using the agar layer method (Dhingra &
Sinclair, 1995). The degree of antagonism of these
antagonists was determined by both cross-culture method
and filter-paper disk method (Dhingra & Sinclair, 1995). Of
these antagonists, three most effective antagonists were
selected based on the degree of inhibition of pathogen and
growth rate of antagonist for in-vitro evaluation studies.
LWIN AND RANAMUKHAARACHCHI / Int. J. Agri. Biol., Vol. 8, No. 5, 2006
658
In-vitro evaluation of potential antagonists. Three
selected antagonists (viz. LR 3, LR 6 & LR 10) and four
commercial antagonistic microorganisms [viz. EM
concentrate, EM instant, EM -fermented plant extract (EM-
FPE) and Bacillus subtilis] were evaluated against the
bacterial wilt pathogen in vitro. The experimental designs
were complete randomized design (CRD) with four
replications. Cross culture method and filter paper disk
method were used in first experiment and second
experiment, respectively. PDA medium was used in both
experiments in order to favor the growth of R.
solanacearum and the potential antagonists.
In-vivo evaluation of potential bio-control agents. The
three selected potential antagonists and five-commercial
bio-control agents [viz., EM instant, EM5 (EM preserved
with vinegar & distilled ethyl alcohol), EM-FPE, Bokashi
(EM fermented compost) and B. subtilis] were evaluated in-
vivo against R. solanacearum in the greenhouse using
susceptible tomato variety Sida. The experimental designs
were complete randomized design (CRD) with five
replications. The temperature and relative humidity of the
greenhouse were set at 30ºC and 80%, respectively in order
to favour the disease development.
In pot experiment I, both antagonists and pathogen
were introduced at the same time. Three selected antagonists
and four commercial bio-control agents were evaluated the
effect of antagonists on the pathogenicity of the causal
organism. Each of the antagonist suspensions was mixed
with pathogen suspension in a screwed cap bottle equally
and allowed for one hour to react each other. After one hour,
15-days-old tomato seedlings were inoculated with the
mixture using root dipping method and then transplanted in
the pots filled with sterilized soil.
In pot experiment II, the antagonists were introduced
one week before the pathogen inoculation. Three selected
antagonists and four commercial bio-control agents were
applied to 21-days-old tomato seedlings growing in separate
pots filled with sterilized soils. Antagonists were applied
regularly up to 6 times at one-week interval. To apply
antagonists, 15 mL of suspension at a concentration of 10
9
cfu/mL of each of the three selected antagonists and 100 mL
of 1:100 diluted solutions from the four commercially
available bio-control agents were used.
In pot experiment III, the antagonists were applied
after the pathogen was established in the soil. Sick pot
method was used to inoculate tomato plants. In this
inoculation method, soils in pots were made to be fully
infested with the R. solanacearum and then the test plants
were transplanted in the pots. In the preparation of sick pots,
all the pots were first filled with bacterial wilt disease
infested soils collected from Pakchong district. The
seedlings of the same susceptible tomato variety were
transplanted in these pots and allowed to grow. When the
plants in the pots died due to bacterial wilt disease, the pots
were re-transplanted with another set of new tomato
seedlings. When the second set of tomato plants also died
due to the disease, the pots containing such soils were
considered as sick pots. There were eight bio-control agents
(viz. three isolated antagonist suspensions, four commercial
antagonist suspensions & Bokashi compost) and a control
treatment evaluated. Soils in the sick pots were treated with
corresponding antagonist suspensions as in pot experiment
2. Bokashi was applied as one time application at 15 days
before transplanting at 1:20 with bokashi: soil ratio. The
control treatment received no antagonists. The 21-days old-
tomato seedlings were transplanted in the pots 15 days after
the first application of antagonists.
Disease severity was recorded 2 to 8 weeks after
inoculation at weekly intervals. A 5 point visual scale, 1
being un-damaged and 5 being completely wilted, was used
for recording disease development as adopted by Kelman
and Person (1961). Standard ANOVA procedure was
applied to test the significance differences in the distance of
inhibition of pathogen by antagonists in the in-vitro
evaluation experiment and Fisher’s protected least
significant difference was used to compare the mean
performance among treatments (Steel & Torrie, 1980). The
visual scores were ranked based on their performance.
IRRI-Stat software was used for data analysis.
RESULTS AND DISCUSSION
Initial screening of potential antagonists. Among the ten
antagonists, LR 3 had the highest distance of inhibition of
the pathogen growth (Table I). LR 10 was the best among
the antagonists isolated from soil. Antagonists isolated from
EM products showed a higher potential for inhibition of the
pathogen (Table I). Filter paper disc method showed
different results to cross-culture method. The antagonistic
ability of LR 3 was lower in the filter paper disk method
compared to that of cross-streak method. It may be due to its
slow growth rate. This could be due to the fact that in the
cross-streak method, antagonists were introduced into the
media two days before the inoculation of pathogen and
hence it had sometime to produce specific toxins or
suppressants by the time pathogen was introduced.
In-vitro evaluation of potential antagonists. EM based
isolates as well as EM products explicitly showed their
ability to inhibit the R. solanacearum with the distance of
inhibition zone (Table II). Antagonistic potential among the
direct EM products showed that EM-FPE was able to
significantly reduce the growth of pathogen and by far
superior to others. When the ranking was done on the
potential of inhibition of the pathogen, EM products were
appeared in the highest. The bacterial wilt disease was also
observed to decrease under EM-FPE in tomatoes and egg
plant (Margarita & Dengel, 2003). Kyan et al. (1999) also
reported that EM contains antagonistic microorganisms and
hence can suppress the plant pathogens. However, the
current study shows the significant role and potential of EM
products in the suppression of R. solanacearum. Growth
inhibition zones on agar media may be due to chemical
BIOLOGICAL CONTROL OF BACTERIAL WILT / Int. J. Agri. Biol., Vol. 8, No. 5, 2006
659
factors (low pH), to antibiotic substances with a broad
spectrum of activity or to more specific bacteriocins, or to
the presence of bacteirophages (Gross & Vidaver, 1990).
In-vivo evaluation of potential bio-control agents. In the
pot experiment I, the disease severity was highest in EM 5
and B. substilis as the control (Table III). EM 4 and LR 3
had the lowest disease condition. EM-FPE, LR 6 and LR 10
had moderate disease conditions. When the antagonists
were introduced prior to pathogen, i.e. in the Experiment II,
all EM-based antagonists suppressed pathogens better than
the rest of the antagonists. In the experiment III, where the
antagonists were introduced after the pathogen, the highest
disease control was found in EM-instant and Bokashi
treatments, while all other antagonists had almost the same
degree of moderate disease severity when compared with
the control (Table III).
The response of antagonists in terms of disease
suppression varied with the antagonist and time of
application. Both EM 5 and B. subtillis have improved their
performance when they were introduced one week before
pathogen inoculation as compared with the introduction of
the antagonists simultaneously with pathogen. But, there
was a slight reduction in the disease suppression when the
antagonists were introduced one week later, using sick pot
technique. The failure B. substilis to suppress the pathogen
in the pot experiment 1 could be due to its less active or
inactive form, as the commercial product of B. substilis is
available in the spore form, which may require a few days in
the soil to activate its action. This has been shown in its
enhanced performance in the pot experiment II and III.
Various Actinomycetes and bacteria including Bacillus
mesenteriacus, B. megaterium, B. subtilis, B. mycoides and
Erwinia have been reported to be active biological control
agents (Kelman, 1953). EM 5 which was not effective in pot
experiment I, was effective in the other experiments. This
may be due to the different application procedure. In the
experiment II and III, 1:100 diluted EM 5 solution was used
but in experiment I, EM 5 concentrated solution was used.
EM 5 contains alcohol and vinegar. It may damage the roots
of the test plant when they were dipped in EM 5 and
pathogen suspension mixture and cause wilt symptom.
Therefore, application method of bio-control agent should
be suitable for antagonist’s action and prior to pathogen
attack.
Bokashi and EM instant showed greater suppression
of bacterial wilt pathogen due to its effective combination of
microorganisms. In addition, Bokashi contained nutrients
(i.e. organic resources) for microbes; thus, EM microbes
could multiply properly and react efficiently. Therefore, it
needs only one application although the others needs 6
times. For these reasons, Bokashi may be reliable bio-
control agent in sustainable management of bacterial wilt.
All bio-control agents tested showed their ability to
reduce the severity of bacterial wilt disease in all pot
experiments. Among the bio-control agents tested, EM-
instant and Bokashi were the best performers in the
Table I. Determination of the degree of antagonism of
ten isolated antagonists
Distance of inhibition zone (mm)
Antagonist
Cross culture
method
Filter paper disc
method
Source
1/
LR1 3.00 0.00 Soil
LR 2 3.00 0.00 Soil
LR 3 11.50 2.12 EM.FPE
2/
LR 4 3.25 0.00 Soil
LR 5 3.25 0.00 Soil
LR 6 5.50 3.37 EM instant
3/
LR 7 5.00 3.62 EM Concentrate
4/
LR 8 2.50 0.62 Soil
LR 9 2.50 0.37 Soil
LR 10 3.50 0.75 Soil
1/ Source indicates where the suspected antagonists were extracted from.
2/ EM-FPE – Effective microorganisms in fermented plant extracts (Kyan
et al., 1999);
3/ EM instant – Effective microorganisms activated with molasses (Kyan
et al., 1999);
4/ EM concentrate – Effective microorganism solutions available (Kyan et
al., 1999);
Table II. Inhibition of R. solanacearum by potential
antagonists in in-vitro studies
Distance of inhibition zone (mm)
Treatments Experiment I
(Crossstreak
method)
Experiment II
(Filter paper disc
method)
Mean
Rank
EM-FPE 18.25
3.87
11.06 1
EM instant 8.00 3.70 5.88 4
EM concentrate 8.25 3.62 5.94 3
B. substilis 1.25
0.00
0.63 7
LR3 12.25 1.75 7.00 2
LR6 5.25
3.25
4.25 5
LR10 3.25
0.00
1.63 6
PROB 0.000 0.000
SE 0.659 0.159
LSD5% 1.939 0.468
CV% 16.300 13.700
Table III. Effect of bio-control agents on the severity of
bacterial wilt disease
Disease severity (Final scoring scale)
Treatments Experiment I
Antagonist
and Pathogen
applied at the
same time
Experiment II
Antagonist
established
before the
pathogen
Experiment III
Antagonist
established
after the
pathogen
Pathogen only (control) 5.0 (4) */
5.0 (8) */
4.6 (7)*/
EM4 instant 1.6 (1)
1.2 (1)
1.4 (1)
EM5
1
5.0 (4)
1.6 (2)
2.8 (4)
EM-FPE 2.2 (3)
1.8 (3)
2.4 (3)
Bacillus substilis 5.0 (4)
2.4 (4)
2.8 (4)
LR3 1.8 (2) 3.0 (6) 3.4 (6)
LR6 2.2 (3)
2.8 (5)
3.2 (5)
LR10 2.2 (3) 3.8 (7)
3.4 (6)
Bokashi - - 1.6 (2)
*/ Value within parenthesis indicates the rank of performance of the
antagonists: 1 being the highest disease suppression (lowest disease
incidence) and increasing the number shows the increasing disease
infestation; 1/ EM5 – EM preserved with vinegar and distilled ethyl
alcohol (Kyan et al., 1999)
LWIN AND RANAMUKHAARACHCHI / Int. J. Agri. Biol., Vol. 8, No. 5, 2006
660
suppression of R. solanacearum. Milagrosa and Balaki
(1999) also reported that the incidence of wilting caused by
P. solanacearum in potato was lower with the application of
Bokashi and EM concentrate alone or combined with
inorganic fertilizer than un-treated control. The results of the
present study confirm that EM contains antagonists against
R. solonacearum and hence, it has the ability to suppress the
bacterial wilt disease. Furthermore, EM has also been
shown to promote the colonization and infection of VAM
(Javaid et al., 2000). This may be another way EM helps
reducing the symptoms of bacterial wilt disease, as it helps
plant to acquire its requirements from soil through the
access of the VAM fungi even when a part of the root
system has been damaged by R. solanacearum.
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(Received 27 January 2006; Accepted 17 June 2006)
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... The saprophytic microorganism that was obtained from spent mushroom media. This method was the same Bokashi technique, were introduced as the best performer in the suppression of R. solanacearum [11]. The application of Bacillus amyloliquefaciens strain S20 alone could control eggplant wilt with an efficacy of 25.3% during a 40 days experiment. ...
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Inhibition of fungi and bacteria by Effective Microorganisms (EM) was investigated by a double-layer plate method. Colony diameters of fungi (Sclerotium rolfsii, Pythium sp., Rhizoctonia solani, Colletotrichum gloeosporioides, Alternaria sp., Thielaviopsis paradoxa, Phytophthora capsici, Aspergillus sp., Fusarium moniliforme and Fusarium oxysporum fs phaseoli) and numbers of bacteria (Xanthomonas campestris pv vesicatoria and Pseudomonas solanacearum) were compared with untreated controls after 24 or 48 hours of incubation. Phytopathogenic fungi and bacteria were generally inhibited by EM in this study. Introduction Suspensions of Effective Microorganisms or EM are mixed cultures of naturally-occurring beneficial microorganisms that can be applied as inoculants to restore nutritionally and biologically degraded soils (Higa, 1994). As a soil inoculant, EM has been reported to decrease the inoculum density of several crop pathogens (Lin, 1991; Higa and Wididana, 1991). Field observations prompted in vitro tests to determine the effect of EM on selected phyiopathogens. Materials and Methods Pure cultures of phytopathogenic fungi and bacteria were used for the in vitro tests. The fungi included: Sclerotium rolfsii, Pythium sp., Rhizoctonia solani, Colletotrichum gloeosporioides, Alternaria sp., Thielaviopsis paradoxa, Phytophthora capsici, Aspergillus sp., Fusarium moniliforme, and Fusarium orysporum fs phaseoli. Pure cultures of bacteria (Xanthomonas campestris pv vesicatoria ENA 4347 and Pseudomonas solanacearum ENA 2720) were also used. Mycelial disks of 0.7 cm diameter were used as inoculum from pure cultures of fungi grown on potato dextrose agar (PDA). Each test petri plate was prepared with one layer of PDA with either 1 or 5 percent concentration of EM, dried for 24 hours, and then covered by another layer of PDA without EM on which the inoculum disks were planted. Control plates were similarly prepared but without addition of EM. Each treatment consisted of two plates with EM and one control plate without EM. In each test, three evaluations of mycelial growth were made, beginning at the edges of the disks, after 24 and 48 hours in a BOD incubator at 30 o C. Each measurement for either growth period was considered as a replication. The bacterial inoculum in an 8.5 percent salt solution was standardized at 600 nm with a colorimeter. For each standard bacterial suspension, 0.1 ml of serial dilutions to 10 -8 were placed into each petri dish with a bottom layer of PDA plus 0.5 or 1 percent of EM supernatant centrifuged at 7500 times gravity for 10 minutes under aseptic conditions. A second layer of dextrose/yeast extract/glutamic acid/peptone agar (DYGS) was added for X.campestris; TTC medium (Kelman) was added for P. solanacearum. Each treatment consisted of two petri dishes (with EM) and one control (without EM) for each dilution. Each test, conducted at four different times, was considered as a replication. Data were obtained for the number of colonies for each serial dilution after 48 hours of incubation at 30 o C.
Characteristics of strains of Pseudomonas sp. and other Phytopathology Effective microorganisms – A holistic technology for humankind
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Potential use of EM for control of phytopathogenic fungi and bacteria Some characteristics involved in bacterial wilt (Pseudomonas solanacearum) resistance in tomato
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Some characteristics involved in bacterial wilt (Pseudomonas solanacearum) resistance in tomato
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